A test method for evaluating the anti-sugar effect of oral care products in vitro

Abstract

The present application belongs to the technical field of efficacy evaluation of oral care products, and discloses a test method for evaluating the anti-sugar effect of oral care products in vitro, and the specific steps are as follows: step one: tooth specimen preparation, provide in-vitro bovine enamel blocks with controllable enamel surface area, polish, ultrasonic cleaning, sterilization, and then use as tooth specimen for standby. The "sugar solution difference method" is created, which breaks through the traditional evaluation framework and realizes mechanism and index dual innovation with "sugar physical barrier" as the target; through the "2min extremely short contact+2-3 days static soaking" double time window protocol, the clinical relevance and high-throughput screening demand are considered, and the test cycle is greatly shortened; the quantitative judgment standard of "anti-sugar ratio >=20% and P<0.05" is established, which provides a basis for "anti-sugar" efficacy claim; the 5mm*5mm standardized in-vitro bovine enamel block model is adopted, which guarantees the experimental reproducibility; the perfect quality control system is built in, which effectively solves the problems of data drift and batch-to-batch non-comparison.

Description

Technical Field

[0001] This invention belongs to the field of oral care product efficacy evaluation technology, specifically a test method for evaluating the anti-glycation effect of oral care products in vitro. Background Technology

[0002] The key step in the development of dental caries is the adhesion and retention of sugars from food on the tooth surface, where they are metabolized by cariogenic bacteria in the mouth to produce acidic substances, leading to demineralization and destruction of the hard tissues of the teeth. Therefore, blocking or reducing the direct contact between sugars and the tooth surface is one of the important strategies for preventing dental caries.

[0003] Currently, the efficacy claims and evaluations of oral care products on the market mostly focus on mechanical cleaning, chemical antibacterial properties, or promoting tooth remineralization through ingredients such as fluoride. With the rise of the "anti-sugar" concept, products claiming to form a barrier on the tooth surface to physically block sugar have begun to appear on the market. However, both the industry and regulatory authorities lack a standardized, quantifiable, and reproducible in vitro testing method to scientifically verify the "anti-sugar" efficacy of such products. Existing in vitro evaluation models, such as microhardness testing, transverse radiometric microscopy (TMR), or pH cycling models, mainly evaluate the "demineralization-remineralization" process or antibacterial effects, and cannot directly and quantitatively reflect the product's physical barrier ability against sugar. These existing technologies suffer from drawbacks such as long development cycles, high costs, and lack of unified standards. Therefore, there is an urgent need to develop an in vitro evaluation method that can quickly construct a stable anti-sugar barrier, is low in cost, has high clinical relevance, and can be used for early formulation screening and patented efficacy claims, and to establish a scientific and reproducible efficacy evaluation system. Summary of the Invention

[0004] The purpose of this invention is to provide an in vitro test method for evaluating the anti-glycation effect of oral care products, in order to solve the problems of lack of standardized, quantitative and rapid detection methods for the "physical barrier of sugar" mechanism of oral care products in the prior art, as well as long test cycles, high costs and poor reproducibility.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a test method for evaluating the anti-glycation effect of oral care products in vitro, the specific steps of which are as follows: Step 1: Tooth specimen preparation. Provide an excised bovine tooth enamel block with controllable enamel surface area. After polishing, ultrasonic cleaning, and sterilization, it is used as a tooth specimen for later use. Step 2: Pretreatment of the sample to be tested. For toothpaste, mix toothpaste and deionized water at a mass ratio of 1:1.6 to form a homogeneous slurry. For non-toothpaste, use directly according to the original or specified ratio in the instructions. Step 3: Sample processing. Immerse the dental specimen in the pre-treated sample solution and soak it continuously at a constant temperature of 37°C for 2 minutes. After rinsing, rinse with clean water for 2 minutes to remove excess sample from the specimen surface. Step 4: Prepare a glucose solution. Weigh a certain amount of glucose, dissolve it in water, and stir until well mixed. After the glucose is fully dissolved, use a glucose reagent kit to detect and record the initial glucose concentration. ; Step 5: Immersion test. Immerse the dental specimens treated in Step 3 in the glucose solution and maintain a constant temperature of 37°C for 2-3 days. After immersion, use a glucose reagent kit to determine the final glucose concentration of the glucose solution. ; Step Six: Calculation and Comparison, calculate the difference in sugar content. A blank control group and a negative control group were set up. Step 7: Statistical analysis. Compare and analyze the ΔC values ​​of the sample group and the blank control group. If the ΔC of the sample group is significantly lower than that of the blank control group, the oral care product is determined to have an anti-glycation effect.

[0006] Preferably, the enamel block of the extracted bovine tooth in step one has an enamel surface area of ​​5mm×5mm, and the enamel surface is free of cracks and defects, and the enamel layer thickness is uniform. It is a healthy extracted bovine tooth enamel block after screening to remove surface impurities and dentin residue.

[0007] Preferably, the specific operations of polishing, ultrasonic cleaning, and sterilization in step one are as follows: the enamel surface of the extracted bovine tooth enamel block is polished using a polishing machine and polishing paste until the enamel surface is smooth and free of scratches; the polished enamel block is placed in an ultrasonic cleaner and ultrasonically cleaned with deionized water for 10-15 minutes; then it is placed in an autoclave and sterilized at 121°C and 0.1MPa for 20 minutes, and then cooled for later use.

[0008] Preferably, the sample to be tested in step two includes toothpaste, mouthwash, oral varnish or candy coating agent, and meets the safety standards for oral care products, and is free from deterioration and odor.

[0009] Preferably, the grouping method of the tooth specimens in step three is as follows: the enamel blocks of the extracted bovine teeth with intact enamel surfaces are randomly divided into a sample group and a blank control group, and at least 3 tooth specimens are set in each group for parallel experiments.

[0010] Preferably, the concentration of the glucose solution in step four is 5% to 10%, and the pH value is stable between 5.5 and 6.5.

[0011] Preferably, in step six, the blank control group uses deionized water instead of the sample solution, and the negative control group is a slurry with the same base material as the sample but without anti-glycation active ingredients. Both the blank control group and the negative control group are tested according to the same operation as in steps two to five.

[0012] Preferably, the criterion for determining "significantly lower" in step seven is: the ΔC value between the sample group and the blank control group is tested by the data statistics method t test, the P value is <0.05, and the anti-glycemic ratio is ≥20%; where P value is the error probability with overall representativeness, and the anti-glycemic ratio = (blank control group ΔC / sample group ΔC) ×100%.

[0013] Preferably, the experiment also includes quality control steps: a standard glucose recovery rate experiment is performed simultaneously for each batch of experiments, with the recovery rate controlled at 95%~105%; enamel blocks are randomly coded and tested using a blind method; the coefficient of variation (CV) of the three parallel data within the experimental group is ≤10%; anti-glycation toothpaste containing 1% Zn-CPC is used as a positive control for calibration every 6 months, and the relative deviation of the retest data between different batches is less than 10%.

[0014] The beneficial effects of this invention are as follows: The innovative "glucose solution difference method" breaks through the traditional evaluation framework, achieving dual innovation in mechanism and indicators by targeting "glucose physical barrier." Through a dual time window protocol of "2-minute ultra-short contact + 2-3 days of static immersion," it balances clinical relevance with high-throughput screening needs, significantly shortening the trial cycle. A quantitative judgment standard of "anti-glucose ratio ≥ 20% and P < 0.05" is established, providing a basis for claims of "anti-glucose" efficacy. A standardized 5mm×5mm extracted bovine tooth enamel block model is used to eliminate the problem of excessive CV caused by inconsistent specific surface areas of traditional powdered or sliced ​​teeth, achieving CV ≤ 10% and inter-laboratory correlation coefficient r ≥ 0.92, ensuring experimental reproducibility. A comprehensive built-in quality control system effectively solves data drift and batch-to-batch incomparability issues, achieving a simple, low-cost, and accurate anti-glucose efficacy evaluation that meets product development and regulatory requirements. Attached Figure Description

[0015] Figure 1 This is a flowchart of the test method of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] like Figure 1 As shown, this embodiment of the invention provides an in vitro test method for evaluating the anti-glycation effect of oral care products. Example 1: Evaluation of the anti-glycation effect of toothpaste products Sample preparation: Toothpaste A was selected as the test sample and mixed with deionized water at a mass ratio of 1:1.6 to form a homogeneous slurry. At the same time, a base toothpaste without anti-glycation active ingredients was prepared as a negative control and mixed with a slurry at the same ratio. Deionized water was used as the blank control. Tooth specimen preparation: Selected bovine teeth with intact enamel were cut into 5mm×5mm enamel blocks, polished, ultrasonically cleaned for 10min, and autoclaved at 121℃ for 20min. The samples were then randomly divided into a sample group, a negative control group, and a blank control group, with 3 tooth specimens in each group. Sample preparation: The three groups of tooth specimens were immersed in toothpaste A slurry, negative control slurry and deionized water respectively, and soaked at 37°C for 2 minutes. After soaking, they were rinsed with water for 2 minutes. Glucose solution preparation: Weigh glucose powder and dissolve it in deionized water to prepare an 8% glucose solution. The initial glucose concentration is then determined using a glucose reagent kit. It is 8.2g / 100mL; Immersion test: The three groups of treated dental specimens were immersed in the above glucose solution at a constant temperature of 37°C for 48 hours. After immersion, the final glucose concentration of the three glucose solutions was measured. The sample group had a concentration of 7.1 g / 100 mL, the negative control group had a concentration of 7.9 g / 100 mL, and the blank control group had a concentration of 8.0 g / 100 mL. Data Calculation and Analysis: ΔC was calculated. For the sample group, ΔC = |8.2 - 7.1| = 1.1 g / 100 mL; for the negative control group, ΔC = |8.2 - 7.9| = 0.3 g / 100 mL; for the blank control group, ΔC = |8.2 - 8.0| = 0.2 g / 100 mL. The anti-glycation ratio was approximately (0.2 / 1.1) × 100% ≈ 27.3%. A t-test showed that the ΔC in the sample group was significantly lower than that in the blank control group (P < 0.05). There was no significant difference in ΔC between the negative control group and the blank control group (P > 0.05). The anti-glycation ratio in the negative control group was approximately (0.2 / 0.3) × 100% ≈ 66.7%. However, since the negative control group did not contain any anti-glycation active ingredients, the difference was not due to the anti-glycation barrier and therefore did not meet the requirements for judging the anti-glycation effect. Results: The anti-glycation rate of toothpaste A sample group was ≥20% and P<0.05, indicating that toothpaste A has an anti-glycation effect.

[0018] The control group setup was comprehensive, with a blank control to eliminate interference from the aqueous phase and a negative control to differentiate the effects of the base material itself and the anti-glycation active ingredients, avoiding misjudgment and ensuring accurate attribution of results. Simultaneously, key parameters were standardized, such as a glaze block size of 5mm×5mm, a treatment time of 2min, and an immersion temperature of 37℃, closely reflecting actual usage scenarios and physiological environments, ensuring strong data reproducibility. Quantitative determination of the anti-glycation effect was achieved through a dual dimension of ΔC value and anti-glycation ratio, combined with t-test statistical analysis, rather than a qualitative description, meeting the requirements of standardized evaluation. Secondly, the anti-glycation mechanism was specifically verified. Although the anti-glycation ratio of the negative control met the standard, it was clearly shown that its difference was unrelated to the anti-glycation barrier, reinforcing the core evaluation logic of "physical sugar barrier" and providing strong support for the method's specificity.

[0019] Example 2: Evaluation of the anti-glycation effect of mouthwash products Sample preparation: Mouthwash B containing 0.2% Zn-CPC was selected as the test sample and used directly according to the original dosage in the instructions. Deionized water was used as the blank control. Tooth specimen preparation: Same as in Example 1, prepare 5mm×5mm ex vivo bovine tooth enamel blocks, randomly divide them into sample group and blank control group, with 3 tooth specimens in each group; Sample preparation: Immerse the two groups of dental specimens in mouthwash B and deionized water respectively, and soak at 37°C for 2 minutes. After soaking, rinse with clean water for 2 minutes. Preparation of glucose solution: Prepare a 6% (w / w) glucose solution and determine the initial glucose concentration. It is 6.3g / 100mL; Immersion test: The two groups of treated tooth specimens were immersed in glucose solution at 37°C for 48 hours. After immersion, the final glucose concentration of the sample groups was measured. The concentration was 5.4 g / 100 mL, while the blank control group had a concentration of 6.1 g / 100 mL. Data calculation and analysis: ΔC of the sample group = |6.3-5.4| = 0.9 g / 100 mL, and ΔC of the blank control group = |6.3-6.1| = 0.2 g / 100 mL; the anti-glycation ratio = (0.2 / 0.9) × 100% ≈ 22.2%. According to the t-test, the ΔC of the sample group was significantly lower than that of the blank control group (P<0.05). Results: The anti-glycation rate of mouthwash B sample group was ≥20% and P<0.05, indicating that mouthwash B has an anti-glycation effect.

[0020] The mouthwash was used directly at the original dosage as per the instructions, without additional dilution or preparation, replicating the application state of daily mouthwash and ensuring a high correlation between the evaluation results and clinical use. The core parameters from Example 1, such as the 5mm×5mm extracted bovine tooth enamel block, 2-minute constant temperature treatment, and 37℃ immersion, were reused to ensure methodological consistency and reproducibility, facilitating horizontal comparisons of effects across different product categories. Only a sample group and a blank control group were set up to focus on the anti-glycation effect of the mouthwash itself, avoiding interference from irrelevant factors. The glucose solution concentration was adapted to the efficacy characteristics of the mouthwash, resulting in significant differences in the test data, which is beneficial for efficacy determination. The scientific verification of the mouthwash's anti-glycation effect was achieved through a quantitative combination of ΔC value, anti-glycation ratio (≥20%), and t-test (P<0.05), which not only meets the unified methodological standards but also highlights the evaluation suitability for this product category, providing a reliable basis for claims of anti-glycation efficacy for mouthwash products.

[0021] Example 3: Evaluation of the anti-glycation effect of oral coating products Sample preparation: Select oral varnish C containing 5% PVM / MA copolymer as the test sample, apply the varnish directly according to the instructions, and use deionized water as the blank control; Tooth specimen preparation: Same as in Example 1, prepare 5mm×5mm ex vivo bovine tooth enamel blocks, randomly divide them into sample group and blank control group, with 3 tooth specimens in each group; Sample preparation: The surface of the tooth specimens in the sample group was evenly coated with oral smear C, and the tooth specimens in the blank control group were immersed in deionized water. Both groups were placed at a constant temperature of 37℃ for 2 minutes. After the sample group was removed, it was gently rinsed with clean water for 2 minutes. Glucose solution preparation: Prepare a 10% glucose solution and determine the initial glucose concentration. It is 10.5g / 100mL; Immersion test: The two groups of treated tooth specimens were immersed in glucose solution at 37°C for 48 hours. After immersion, the final glucose concentration of the sample groups was measured. The concentration was 9.2 g / 100 mL, while the blank control group had a concentration of 10.3 g / 100 mL. Data calculation and analysis: ΔC for the sample group = |10.5-9.2| = 1.3 g / 100 mL, and ΔC for the blank control group = |10.5-10.3| = 0.2 g / 100 mL; the anti-glycation ratio = (0.2 / 1.3) × 100% ≈ 23.1%. According to the t-test, the ΔC of the sample group was significantly lower than that of the blank control group (P<0.05). Results: The antiglycation rate of oral varnish C sample group was ≥20% and P<0.05, indicating that oral varnish C has an antiglycation effect.

[0022] The sample processing adopts a "uniform application" method, which aligns with the actual usage logic of coating products, avoiding misjudgments of efficacy due to improper processing and ensuring a high correlation between evaluation results and actual product application effects. Continuing the standardized core system, it reuses unified parameters such as 5mm×5mm excised bovine tooth enamel blocks, 2-minute constant temperature placement, and 37℃ immersion, ensuring consistency with evaluation methods for toothpaste and mouthwash products and facilitating cross-category effect comparisons. The ΔC value difference between the sample group and the blank control group is clear, improving the sensitivity and accuracy of efficacy determination. Simultaneously, using ΔC value, ≥20% anti-glycation ratio, and P<0.05 statistical results as core criteria, it adheres to the unified standards of the overall experimental method while specifically adapting to the dosage form characteristics of coating products, providing a reliable and reusable standardized example for verifying the anti-glycation efficacy of this product category.

[0023] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0024] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A test method for evaluating the anti-glycation effect of oral care products in vitro, characterized in that, The specific steps are as follows: Step 1: Tooth specimen preparation. Provide an excised bovine tooth enamel block with controllable enamel surface area. After polishing, ultrasonic cleaning, and sterilization, it is used as a tooth specimen for later use. Step 2: Pretreatment of the sample to be tested. For toothpaste, mix toothpaste and deionized water at a mass ratio of 1:1.6 to form a homogeneous slurry. For non-toothpaste, use directly according to the original or specified ratio in the instructions. Step 3: Sample processing. Immerse the dental specimen in the pre-treated sample solution and soak it continuously at a constant temperature of 37°C for 2 minutes. After rinsing, rinse with clean water for 2 minutes to remove excess sample from the specimen surface. Step 4: Prepare a glucose solution. Weigh a certain amount of glucose, dissolve it in water, and stir until well mixed. After the glucose is fully dissolved, use a glucose reagent kit to detect and record the initial glucose concentration. ; Step 5: Immersion test. Immerse the dental specimens treated in Step 3 in the glucose solution and maintain a constant temperature of 37°C for 2-3 days. After immersion, use a glucose reagent kit to determine the final glucose concentration of the glucose solution. ; Step Six: Calculation and Comparison, calculate the difference in sugar content. A blank control group and a negative control group were set up. Step 7: Statistical analysis. Compare and analyze the ΔC values ​​of the sample group and the blank control group. If the ΔC of the sample group is significantly lower than that of the blank control group, the oral care product is determined to have an anti-glycation effect.

2. The test method for evaluating the anti-glycation effect of oral care products in vitro according to claim 1, characterized in that: The enamel block of the extracted bovine tooth mentioned in step one has an enamel surface area of ​​5mm×5mm, and the enamel surface is free of cracks and defects, and the enamel layer thickness is uniform. After screening, healthy extracted bovine tooth enamel blocks with surface impurities and dentin residue are removed.

3. The test method for evaluating the anti-glycation effect of oral care products in vitro according to claim 1, characterized in that: The specific operations for polishing, ultrasonic cleaning, and sterilization described in step one are as follows: Use a polishing machine and polishing paste to polish the enamel surface of the extracted bovine tooth enamel block until the enamel surface is smooth and free of scratches; place the polished enamel block in an ultrasonic cleaner and ultrasonically clean it with deionized water for 10-15 minutes; then place it in an autoclave and sterilize it at 121℃ and 0.1MPa for 20 minutes, and then cool it for later use.

4. The test method for evaluating the anti-glycation effect of oral care products in vitro according to claim 1, characterized in that: The samples to be tested in step two include toothpaste, mouthwash, oral varnish or candy coating agent, and must meet the safety standards for oral care products and be free from deterioration and odor.

5. The test method for evaluating the anti-glycation effect of oral care products in vitro according to claim 1, characterized in that: The grouping method for the tooth specimens in step three is as follows: the enamel blocks of intact bovine teeth extracted from the body are randomly divided into a sample group and a blank control group, with at least 3 tooth specimens in each group for parallel testing.

6. The test method for evaluating the anti-glycation effect of oral care products in vitro according to claim 1, characterized in that: The concentration of the glucose solution mentioned in step four is 5% to 10%, and the pH value is stable between 5.5 and 6.

5.

7. The test method for evaluating the anti-glycation effect of oral care products in vitro according to claim 1, characterized in that: In step six, the blank control group uses deionized water instead of the sample solution, and the negative control group is a slurry with the same base material as the sample but without anti-glycation active ingredients. Both the blank control group and the negative control group are tested according to the same procedures as in steps two to five.

8. The test method for evaluating the anti-glycation effect of oral care products in vitro according to claim 1, characterized in that: The criteria for determining a significantly lower value as described in step seven are: the ΔC value between the sample group and the blank control group, tested using the statistical method t-test, has a P-value < 0.05, and the anti-glycemic ratio is ≥ 20%; where P-value represents the probability of error with overall representativeness, and the anti-glycemic ratio = (ΔC of the blank control group / ΔC of the sample group) × 100%.

9. The test method for evaluating the anti-glycation effect of oral care products in vitro according to claim 1, characterized in that: It also includes quality control steps: standard glucose recovery experiments are conducted simultaneously for each batch of experiments, with the recovery rate controlled at 95%~105%; enamel blocks are randomly coded and tested using a blind method; the coefficient of variation (CV) of the three parallel data within the experimental group is ≤10%; anti-glycation toothpaste containing 1% Zn-CPC is used as a positive control for calibration every 6 months, and the relative deviation of the retest data between different batches is less than 10%.

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